Despite decades of research, the morbidity and mortality of necrotizing enterocolitis (NEC) remain unchanged due to a fundamental gap in our understanding of how to protect the intestines from this disease. Evidence suggests that an immature enteric nervous system (ENS) may predispose prematures to NEC. We have demonstrated protracted ENS abnormalities in human and rodent NEC-injured intestine that persist long after apparent recovery from the acute disease. Our preliminary data demonstrate that heparin-binding EGF-like growth factor (HB-EGF) and stem cell (SC) transplantation act collectively to protect the intestines and the ENS from experimental NEC. The long-term goal of our work is to identify novel prophylactic and therapeutic strategies to protect neonates from NEC. The overall objective of the current renewal application is to identify key elements of the interaction of HB-EGF with SC that may be utilized to improve protection of the intestines from injury. Our central hypothesis is that the ability of HB-EGF to protect SC plays a major role in it intestinal cytoprotective effects, and that the beneficial effects of SC are mediated by SC-secreted exosomes. Exosomes are nanovesicles that are secreted by many cell types and which contain a complex mixture of microRNAs, mRNAs and proteins that reflect the transcriptional and/or translational activity of their producer cells. We will objectively test our central hypothesis by pursuing the following specific aims: Aim 1) to determine which biological source of stem cells is most effective in protecting the intestine and the ENS from injury during NEC. Aim 2) to establish the exosome dependency of stem cell therapy and the beneficial effects of exosomal HB-EGF. Aim 3) to determine whether the microRNA content of circulating exosomes can be used as biomarkers of NEC. The significance of the proposed research is that it will lead to a better understanding of the effects of HB- EGF and SC on protection of the intestines and the ENS, allowing us to best design potential clinical HB-EGF- and SC-based therapies to treat NEC. The positive impact of our studies will be development of improved therapeutic interventions for patients at risk of developing NEC and fundamental advances in understanding growth factor-stem cell interactions in the intestines. The proposed research is innovative because we seek to utilize novel experimental therapies designed to protect and regenerate the intestine and the ENS through administration of an intestinal cytoprotective growth factor in conjunction with stem cells, and because it will further investigate the novel observation that HB-EGF acts as a pro-motility agent.